The present invention relates to a method of plasmid recovery and an apparatus for doing so. More particularly, it relates to a method of plasmid preparation and the devices for accomplishing the method.
The conventional method for recovering plasmid from a bacterial lysate after alkaline lysis is commonly known as a bind, wash, and elute process. This process recovers plasmid from a cleared lysate by binding to a glass fiber filter in the presence of a chaotropic agent, such as potassium iodide. This chaotropic agent causes the plasmid to bind to the glass fibers, while most of the other cell constituents pass through. The glass fiber filter is then washed with ethanol (70% or higher by weight) to remove the chaotropic agent. Excess ethanol is removed from the underside of the filter plate by blotting, centrifugation or extensive vacuum drying. The plasmid is then eluted from the glass fibers using water or a low salt buffer.
This method has many drawbacks. First, it introduces a contaminant (ethanol) to the system, which is difficult to fully remove. The residual ethanol may adversely affect the plasmids or the tests performed on them. Additionally, this is a time consuming process and requires many sequential steps. Further, the capacity of the glass fibers to bind the plasmids is limited, making the binding inefficient. Likewise, the elution from the glass is not complete. Some plasmid has been found to irreversibly bind to the glass. In sum, the recovery of the plasmids often is less then 80%, sometimes less then 70% of the available plasmids.
The present invention provides a better method and apparatus for recovering plasmids or other circular DNA which eliminates the introduction of new contaminants, provides for higher plasmid recovery rates with higher purity and which is much faster than the current process.
The present invention provides a method for rupturing cells, and then filtering the ruptured cells through one or more microfiltration (MF) or coarse filtration membranes. Most cellular debris is removed by the membranes(s) and the remainder is then filtered through an ultrafiltration (UF) membrane so that the plasmids or other DNA is retained on the upper surface of the UF membrane, where it is recovered.
The process may use either centrifugation or a constant pressure differential (positive or negative) to effect both the MF or coarse filtration and UF filtration steps. It is preferred that the process use a constant pressure differential for both steps, and more particularly, it is preferred that a negative constant pressure differential (vacuum) be used in both steps.
Additionally, an apparatus for effecting this process is disclosed. It is comprised of an upper filter plate containing one or more wells, each well(s) having a MF or coarse filtration membrane located within it, preferably adjacent the bottom of the well(s). The upper plate has a connection to a supply for a constant pressure differential. A lower plate is provided which contains one or more wells, each well(s) having an UF membrane located with it, preferably adjacent to the bottom of the wells. The lower plate has a connection to a supply of negative constant pressure differential. Below the lower plate is a liquid waste collector or drain.
It is an object of the present invention to provide a process for recovering plasmids or other DNA comprising the steps of:
(a) disrupting the cell walls sufficiently to free the cellular components, in particular plasmids and other DNA;
(b) filtering the cellular components through one or more microfiltration or coarse filtration membranes or combinations of the two and collecting the filtrate;
(c) filtering the filtrate of (b) through one or more ultrafiltration membranes so as to leave the plasmids or other DNA as a retentate on the upper surface of one or more ultrafiltration membranes; and
(d) recovering the plasmids or other DNA from the upper surface of the ultrafiltration membrane.
It is a further object of the present invention to provide a process for recovering plasmids or other DNA comprising the steps of:
(a) disrupting the cell walls sufficiently to free the cellular components, in particular plasmids or other DNA;
(b) filtering the cellular components through one or more microfiltration or coarse filtration membranes or combinations of the two using a positive pressure to drive the filtration and collecting the filtrate;
(c) filtering the filtrate of (b) through one or more ultrafiltration membranes using a negative pressure to drive the filtration so as to leave the plasmids or other DNA as a retentate on the upper surface of one or more ultrafiltration membranes; and
(d) recovering the plasmids or other DNA from the upper surface of the ultrafiltration membrane.
It is a further object of the present invention to provide a process for recovering plasmids or other DNA comprising the steps of:
(a) disrupting the cell walls sufficiently to free the cellular components, in particular plasmids or other DNA;
(b) filtering the cellular components through one or more microfiltration or coarse filtration membranes or combinations of the two using a positive pressure to drive the filtration and collecting the filtrate;
(c) filtering the filtrate of (b) through one or more ultrafiltration membranes using a positive pressure to drive the filtration so as to leave the plasmids or other DNA as a retentate on the upper surface of one or more ultrafiltration membranes; and
(d) recovering the plasmids or other DNA from the upper surface of the ultrafiltration membrane.
It is a further object of the present invention to provide a process for recovering plasmids or other DNA comprising the steps of:
(a) disrupting the cell walls sufficiently to free the cellular components, in particular plasmids or other DNA;
(b) filtering the cellular components through one or more microfiltration or coarse filtration membranes or combinations of the two using a negative pressure to drive the filtration and collecting the filtrate;
(c) filtering the filtrate of (b) through one or more ultrafiltration membranes using a positive pressure to drive the filtration so as to leave the plasmids or other DNA as a retentate on the upper surface of one or more ultrafiltration membranes; and
(d) recovering the plasmids or other DNA from the upper surface of the ultrafiltration membrane.
It is a further object of the presentation to provide a process for recovering plasmids or other DNA comprising the steps of:
(a) disrupting the cell walls sufficiently to free the cellular components, in particular plasmids or other DNA;
(b) filtering the cellular components through one or more microfiltration or coarse filtration membranes or combinations of the two using a negative pressure to drive the filtration and collecting the filtrate;
(c) filtering the filtrate of (b) through one or more ultrafiltration membranes using a negative pressure to drive the filtration so as to leave the plasmids or other DNA as a retentate on the upper surface of one or more ultrafiltration membranes; and
(d) recovering the plasmids or other DNA from the upper surface of the ultrafiltration membrane.
It is another object of the present invention to provide an apparatus for recovering plasmids or other DNA comprising an upper filter plate having one or more microfiltration or coarse filtration membranes and a lower filter plate having one or more ultrafiltration membranes, the upper filter plate being located above and adjacent to the lower filter plate.
These and other objects of the present invention will become clear from the description of the invention and the claims.